Immune responses against donor (i.e., “non-self” or “allogeneic”) antigens are the primary cause of rejection of the transplanted cells, resulting in graft failure. To date, the primary strategies for avoiding rejection have been to minimize antigenic differences between donor and recipient by matching Human Leukocyte Antigens (HLA; also known as Major Histocompatibility (MHC) antigens) and by subjecting the transplant recipient to potent immunosuppression.
HLA antigens are encoded by several gene loci; of these, the most important for graft survival are the HLA Class I antigens A and B (see FIG. 1), and the Class II antigen (FIG. 2) DR. An HLA matching effect was significantly associated with HLA-A, B, and DR match in kidney transplantation and has an overall significant effect for graft survival and/or rejection in pancreas, heart, lung and bone marrow transplantation. In liver transplants, matching was associated with less rejection but was also associated with reoccurrence of disease. Thus, the extent to which the HLA-A, B, and DR loci antigens are matched is currently one of the most significant parameters affecting outcome in most solid organ and bone marrow transplants.
Kidney Transplants
Many factors are associated with increased renal transplant survival, one of which is living-related donors, who will generally have more closely matched set of HLA antigens than unrelated donors. Patients cannot always benefit from a living-related transplant compared to a cadaveric renal transplant if risk factors were increased in a living donor transplant [7]. For example, graft survival in recipients 18 to 59 years of age with a live 50-year-old kidney donor is comparable or decreased compared with a cadaveric renal transplant if the cadaveric donor is much younger or has fewer HLA mismatches. On the other hand, cadaver renal transplants in recipients over 60 never provide better outcome than living-related transplants. These are significant observations since live donor transplantation now comprises more than 40% of all kidney transplantation and will continue to be a large part of renal transplantation [8] as long as the number of cadaver donors remains constant, as it has through the last ten years. In either case, mismatching of HLA antigens incurs immune responses that reduce graft survival.
It has been shown that race is one of the risk factors associated with renal transplant results. Asians have very high graft survival rates and the question has been raised regarding whether or not matching has an effect on results. However, Opelz [9] shows that HLA matching does have an additive effect on graft survival in Asians.
HLA-DP allele matching in renal transplants was known to have a significant effect on renal graft survival. Examination of regraft recipients which were all one allele match, showed that immunogenetic epitopes were important to match [10]. These results corroborate the importance of immunogenicity, noting that alleles may not always be immunogenic. This is quite similar to the result found in bone marrow transplantation with “alleles” solely defined by molecular techniques [47, 48, 49].
There has been an ongoing contention that new immunosuppressive drugs improve graft survival rates, which proscribes the need for HLA matching [1, 2, 3]. In an analysis [1] with over 52,000 transplants from 1978 through 2000, the transplants were broken into three eras: the first was—1978 to 1984, prior to calcinurin inhibitor drugs cyclosporin A (CsA) and FK-506; the CsA era, 1987 through 1994; and after extensive use of FK-506 from 1995 to 2000. Results were shown with each match grade and 10-year graft survival. In every instance, notwithstanding the difference in immunosuppressive drugs utilized, the 10-year graft survival difference between zero mismatch and six antigens mismatch was 18%.
This consistent difference was found even though transplantation has changed dramatically through the years. That is, one year graft survivals were 58% in 1978-84 as compared to 86% in the most recent (1995 to 2000) era. In addition to major changes in immunosuppression, there has been a policy for prospective matching of zero HLA-A, B, DR donor mismatched patients in the USA. In the '78-'84 era, the number of zero mismatched patients was two-hundred eleven (2%) and in the most recent era, 1995 to 2000, 6,279 (14%) HLA zero mismatched patients were transplanted. From the large difference between zero and one mismatch, it is clear that even one antigen mismatch was enough for the immune system to respond and react vigorously. Also, there was an additive increase in graft survival for each mismatched antigen, with the largest increase occurring from one to zero.
Renal transplant half-life data shows an advantage to HLA matching at all match grades, particularly at zero mismatch. Half-life increases as match increases and also increases with better immunosuppression. Furthermore, both the matching and immunosuppressive effects are additive. New changes in kidney allocation by United Network for Organ Sharing (UNOS) eliminate HLA-B, DR matching points in an attempt to decrease the disadvantage to minorities and those with rare HLA types. Because of the contribution of each HLA A, B, DR match, this algorithm change will reduce graft survival in transplant recipients.
Pancreas Transplants
Overall, graft survival in pancreas transplants is lower than in kidney transplants. One possibility for these discrepant outcomes as compared to kidneys, results from diabetic complications. Functional pancreas graft survival, excluding patients that die with a functioning graft, compared to functional kidney graft survival gives similar results, presumably because there is a higher death rate due to diabetic complications.
The role of HLA matching in pancreas transplantation is somewhat controversial. Two recent investigations have reported little HLA matching effect since one year and 5-year graft survival of matched and non-matched were similar [12, 13]; however, the number of transplants examined was quite small. In fact, it has been shown in the international pancreas registry [11] that pancreas after kidney, or pancreas transplant alone, has an HLA Class I matching effect such that if one antigen is matched at either HLA-A or B loci, there are significant one-year pancreas survival rates of 85% and 74% respectively, versus 70% and 60% for the non-matched. Furthermore, poorly matched patients have shown significant increases in rejection [14].
In pancreas transplant recipients, autoantibody to each of islet cells and glutamic acid carboxylase was found in patients with graft complications [15]. Furthermore, pancreas transplant recipients with rejection have anti-HLA Class I and Class II antibodies developing post-transplant [16]. HLA Class II antibodies were significantly associated with risk of chronic allograft rejection [16].
Liver Transplantation
Although the influence of matching on outcome of liver graft survival is controversial [17, 18], HLA-A and B matching is significantly associated with lower graft rejection [18, 19], but there was no beneficial effect of matching HLA-DR. Furthermore, if HLA-DR was matched in liver transplant patients, certain disease conditions increase or may recur; therefore, HLA DR played a role in the etiology of the disease [20].
In hepatitis-B infected liver transplant recipients, there was significant graft survival improvement in patients with HLA-A and B compatibilities but not DR [21]. Specificities of HLA Class II antigens were associated with reduced risk for viral infections, that is HLA-DR 11 and HLA-DQ-3 antigen occurrence in the recipient candidates is a reduced risk for hepatitis C infection [22]. Also, the frequency of HLA-DQ-0302 (an allele of DQ-3) was significantly higher in liver graft recipients with acute rejection [23]. Preformed HLA antibodies have not been associated with graft rejection problems such that positive crossmatch is not necessarily a contra-indication for liver transplantation. This is partly due to the liver's large organ mass, its consequent ability to absorb or “sponge up” the antibodies without any deleterious consequences. In small size grafts with positive flow crossmatches, rejection episodes and organ failure have been noted in four cases [24].
Heart Transplantation
Heart transplant outcomes show a significant correlation to HLA matching [25-28]. In zero mismatches, there is a decrease in risk ratio as the number of matches increases. In one study [28] there seems to be an association between the different HLA loci matches and the risk ratio. That is, HLA-A locus matching may be potentiating antigen presentation in heart recipients, such that there was lower graft survival in HLA-A locus matched transplants. Recent studies [25, 26] have shown similar results. Again, because there is no prospective heart transplant matching system in place, large numbers of well-matched transplants are not available for analysis.
Using left ventricular assist devices has been an important advance as a bridge to cardiac transplantation. However, sensitization to HLA antigens occurs in patients using the left ventricular system devices [29, 30]. In these sensitized patients there was increased acute rejection, but not a decrease in graft survival [29, 30], when compared to non-sensitized patients. A major finding with left ventricular assist devices has been that even though antibody is produced, it can be controlled through plasmapheresis and also through treatment with IVIG and/or Retuxin, an anti CD20 antibody. In other studies, HLA antibodies were associated with a significantly higher incidence of graft rejection episodes and graft loss in heart transplant recipients [31, 32]. This may be reconciled with the above studies [29, 30] in that antibodies are demonstrated by two methods—complement dependent and flow cytometry panel reactive antibodies. The latter shows a much higher correlation to graft loss and rejection, whereas the former shows less correlation because of the relative insensitivity of the test.
HLA-G is an HLA Class I antigen associated with the trophoblast, which inhibits cellular immunity during pregnancy. In cardiac transplants expressing HLA-G, acute rejection was significantly lower [33], and chronic rejection was not found. IL-2 lymphokine polymorphisms may also show an important significant correlation with outcomes [34]. Furthermore, there was a complex interaction with HLA-DR matching, such that patients with IL-2 polymorphisms significantly associated with acute rejection had a better outcome if they were HLA-DR matched.
Lung Transplantation
In lung transplantation, Bronchiolitis Obliterans Syndrome (BOS) is a manifestation of the rejection and graft loss process. The test for BOS is based on forced air volume declining to less than 80% of baseline as well as fibrosis and death of airway epithelial cells. An association was observed between the HLA matching and BOS [35, 36, 37, 38, 39], with different effects noted for HLA Class I and Class II [35, 36, 37, 38]; that is, there was a trend of poorly matched HLA-DR and HLA-A recipients having a significant risk for BOS. Upon examination of the total number of HLA-A, B, and DR mismatches [36], there is a significant association was observed with the appearance of BOS, but not with 5-year mortality rates, which seemed to correlate with repeat regrafting, congenital heart disease, and recipient age. Again, HLA-DR is associated with the appearance of BOS and also graft loss [37, 38]. One long-term study showed significant association of HLA-A and B mismatches, with the occurrence of BOS [39]. In this long-term study, the HLA-A, B mismatches were strongly associated (P=0.002) with the occurrence of BOS at 4 years, and none of the other factors, such as donor/recipient age, ischemia time, pulmonary bypass, episodes of acute rejection, CMV, pneumonitis, or CsA trough levels, had any long-term consequences associated with the occurrence of BOS or graft survival [39]. Matching trends were uncertain since there were not a large number of well-matched patients in these lung transplant studies.
With regard to the BOS post-transplant and an association with HLA antibodies, several papers [40, 41, 42, 43, 44] show the correlation of HLA antibody with the occurrence of BOS. Furthermore, monoclonal antibodies to HLA common antigens stimulated in vitro airway epithelial cell proliferation, which is an initiation event in the development of BOS [40]. Patients who have post-transplant antibody, 90% of which is developed against HLA Class II antigens, showed a significant (P=0.005) association for the development of BOS [41]. Furthermore, in a small study, BOS and death was reported during the follow-up period in four patients who had pre-formed anti-HLA antibodies. Albeit, this was a small study, Class II antibody specificities may have played an important role in both the development of BOS and the chronic rejection. It is an object of an embodiment of the invention herein to avoid sensitization prior to transplant because of the occurrence of BOS and the ultimate death of these recipients.
The mechanism by which the development of BOS occurs with HLA antibody is only partially understood [43, 44]. Antibodies can initiate the cascade of proliferation and formation of growth factor leading to fibrosis. Furthermore, apoptosis is also associated with an anti-HLA antibody binding to the airway epithelial cells [44].
A clear T-cell immune response to HLA Class I and Class II antigens was associated with patients who have BOS [45]. Lymphokine polymorphisms in BOS patients showed significant correlation to high producing IL-6 and interferon gamma, the latter cause de novo synthesis HLA Class II antigens and, therefore, are linked to the association between HLA Class II antibody and the appearance of BOS [46].
Bone Marrow Transplantation
Bone marrow transplantation is distinguished from solid organ transplants since the organ being transplanted is the hematopoietic and immune system. Therefore, in recipients, the host bone marrow is ablated or killed so that it does not react to the donor bone marrow. Furthermore, if there is any recipient HLA antigen mismatching to the donor, graft vs host disease can occur which can have severe complications [47]. Therefore recipient mismatches are relevant whereas in solid organ transplants one is concerned with the donor mismatches. For these reasons, HLA perfectly matched bone marrow transplantation is desirable. Small molecular genetic differences are found to cause bone marrow transplant rejection [48]. These HLA differences can basically be divided into those serologically defined and those that are molecularly defined. In the former case, there may be more than one molecularly based epitope difference in the molecule, whereas, in the latter, a clearly defined single sequence difference is associated with the molecular antigen. It has recently been shown that if one mismatches the serological antigens, this is enough to increase the probability of graft failure [49], however, a single HLA molecular mismatched antigen does not result in increased graft failure. Since HLA is remarkably polymorphic with more than 220, 460, 110, and 360 molecularly defined epitopes for HLA-A, B, C, and DR respectively, the manner in which antigens are defined, and which antigens are mismatched plays an important role in future bone marrow transplantations and in finding a match.
Treatment protocols and bone marrow graft survivals are quite different, depending on the disease of patients being transplanted. Results vary depending upon leukemia type, solid tumor, or syndrome that is treated [50, 51].
Matching HLA-C locus (a generally weakly expressed Class I antigen) may be important regarding the activation of natural killer cells, since C locus serves as a killing receptor inhibitor [52]. It has been shown that one mismatch at the C locus, i.e. the absence of one killing receptor inhibitor, is enough to activate an allogeneic NK cell killing.
As in solid organ transplants above, the association of genetic polymorphism of interferon gamma and IL6 high producing phenotypes has recently been associated with graft vs host disease [53].
Cornea Grafts
Cornea grafts have not been associated with the HLA matching effect [54]. Recently, however, this has been challenged [55, 56] by results showing HLA matching especially HLA-DR, to be significantly correlated to outcome. HLA antibodies have been a significant problem in corneal grafts when observed, and are correlated with poor outcomes [54].
Immune Reactivity to HLA
An in-vitro correlate, i.e. assay, for humoral and cellular response is the formation of HLA specific antibody. That is, with the IgG immunoglobulin formation, there is the requirement of CD4 T cell activation with concomitant Class II antigen presentation. Therefore, the presence of IgG (as demonstrated with panel reactive antibodies) implicates CD4 T-cells and B-cells directly. It is envisioned herein that rejection and/or graft loss as a consequence of the immune response to HLA antigens would usually, if not always, show HLA antibody as an in vitro correlate.
However, until recently, an association of HLA antibody and transplant rejection and loss was not clear. With the advent of new, more sensitive IgG specific techniques such as flow PRA [57] and ELISA [58] in renal transplants [59], it has recently been shown that 96% of all patients who had rejected kidneys had HLA antibody present post-rejection [60]. It has been long known that preformed donor specific HLA antibodies in kidney transplant recipients can be utilized to determine the risk of hyperacute rejection [60-62].
HLA antibodies in renal transplants are associated with acute rejection [64-66]. Furthermore, there seems to be an association between HLA antibody, specifically Class II antibody [16, 41, 66] and chronic rejection, as recently reviewed [67].